Project Summary/Abstract This project seeks to explore the brain circuitry that relays the signal for CO2 arousal to the forebrain during obstructive sleep apnea (OSA). Our previous studies have implicated the external lateral parabrachial subnucleus (PBel) as a key site for relaying signals to the forebrain that cause arousal during brief exposure to high CO2 levels, such as occur during OSA. Those studies further identified key targets of the PBel which may relay this arousal: the supramammillary nucleus (SUM); orexin fields in the lateral hypothalamus (LH); substantia innominata (SI) just ventral to the anterior pole of the globus pallidus; central nucleus of the amygdala (CeA); and bed nucleus of the stria terminalis (BST). In Aim 1, we propose to inject the PBel on one side of the brain with an adeno-associated viral vector (AAV) expressing the genes for Archaerhodopsin TP009 (ArchT) and green fluorescent protein (GFP) in a Cre-dependent manner (AAV-ArchT-GFP) in mice that express Cre recombinase under the CGRP promoter (a gene that is selectively expressed in the PBel). The PBel on the other side of the brain will be genetically lesioned with an AAV that expresses the lethal diphtheria toxin A in a Cre-dependent manner. We will then determine the effect on CO2 arousal of optogenetic inhibition of either the remaining PBel or its key target regions in the forebrain. Studies from other labs have indicated that both the serotonin system and locus coeruleus (LC) modulate CO2 arousal, and we hypothesize that this occurs via interactions of these systems with either the PBel itself, or with its targets. In Aim 2, we will examine the serotonin system by injecting either the dorsal or medullary raphe (or both) in serotonin transporter-Cre (Sert-Cre) mice with AAVs that code for Cre-dependent expression of the hM4Di-mCherry inhibitory receptor. We will then examine the effect on CO2 arousal of inhibiting the serotonin neurons with systemic injection of hM4Di ligand, clozapine-N-oxide. To determine the site at which these neurons act, we will inject the raphe site(s) that affect CO2 arousal with the AAV-ArchT-GFP vector and genetically lesion the PBel with AAV-DTA on one side of the brain, and then attempt to block CO2 arousal by optogenetically inhibiting the key raphe site or its terminals in the remaining PBel or the targets of the PBel. In Aim 3, we will do a parallel set of experiments for the LC to study the noradrenergic contribution to CO2 arousal. Finally, in Aim 4, we will inject the PBel with AAV-ChannelRhodopsin2-mCherry, and in brain slices we will determine the effects of optogenetically activating PBel terminals on patch clamp recordings from different classes of neurons in its target areas, and the effects on this activation of serotonin or norepinephrine or their antagonists. These experiments will identify key components of the chemical circuitry that underlies CO2 EEG arousal, and provide the basis for pharmacological attempts to intervene in this process.